Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 160-166
ISSN: 2319-7706 Volume 3 Number 1 (2014) pp. 160-166
http://www.ijcmas.com
Original Research Article
Preventive and Curative Role of Belladonna 200 Against Japanese
Encephalitis Virus Infection in Adult Mice
M.Sengupta1, B Bandyopadhyay2 and S. Das3*
1
West Bengal University of Health Sciences, DD-36, Sector-1, Salt Lake City,
Kolkata-64, India
2
Department of Microbiology, Virology Unit, School of Tropical Medicine,
Kolkata-700073, India
3
Department of Microbiology, Peerless Hospital and B.K. Roy Research Centre,
Kolkata-700094, India
*Corresponding author
ABSTRACT
Keywords
Japanese
encephalitis;
Belladonna200;
SA14-14-2 JE
Vaccine.
About 3 billion people are now at risk of Japanese encephalitis (JE) having its
focus in South East Asian and Oceanian countries. There is no treatment of this
disease and vaccination is still not adopted as a national program by most countries
endemic to the disease. Thus this study was aimed to see possible preventive and
curative effect of Belladonna-200 against JE. This is an ultra-diluted
homeopathic medicine without any known side effect and suitable for easy oral
administration in inhabitants of remote villages and forest areas in the affected
countries. The experiment was done in adult mice with adapted virulent Nakayama
strain JE virus by a novel passage technique developed by us. In the control group
43.4% mice died after the adapted JE virus challenge. In the vaccinated group with
SA14-14-2 JE vaccination all mice remained alive after the virus challenge and in
the Belladonna-200 treated group as preventive, curative and simultaneously
treated options 13.6%, 22.8% and 50.0% mice died respectively after the
challenge. The difference of outcome with SA14-14-2 JE vaccinated group and
Belladonna-200 preventive treatment was not statistically significant.
Introduction
The burden of Japanese encephalitis (JE)
is still very high despite better vaccine
coverage (Campbell et al., 2011). JE is
now endemic in 24 Asian and Oceanian
countries with an estimated 67,900 JE
cases occurring annually (Campbell et al.,
2011) with about 3 billion population is at
160
risk. JE virus belong to the genus
Flavivirus under the family Flaviviridae
and is classified phylogenetically (Uchil,
2001; Li, 2011; Mohammed, 2011) into
five genotypes (G1-G5) and the mode of
JE virus dispersal study throughout the
Asian countries using Bayesian Markov
Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 160-166
Chain Monte Carlo (MCMC) simulations
indicated its potential for introduction into
non-endemic areas ( Gao et al., 2013). JE
is a mosquito borne disease with a 20-30%
case fatality rate and neurologic sequel in
30-50% of survivors ((Campbell et al.,
2011). The state of JE surveillance and
immunization program in 2012, showed
that there was no immunization program
in 12 out of 24 countries and national
program was present only in five countries
among them. Again different types of
vaccinations were used in different
countries, thus in that study, live
attenuated SA 14-14-2 JE vaccine (LAV)
was used in five countries, inactivated
mouse brain- derived JE vaccine (MB)
was used in six countries and inactivated
Vero cell culture derived JE vaccine (VC)
was used in two countries. In China they
used both LAV and VC. Thus it is a long
way to improve awareness of JE disease
and implementation of a national JE
vaccine program in all the endemic
countries. In our previous studies
(Bandyopadhyay, 2010; Bandyopadhyay,
2011), we observed that simple
homeopathic medicines like Belladonna
200 could prevent JE virus infection in
Swiss albino mice and in chorio-allantoic
membrane of chick. Thus in this study we
compared preventive efficacy of this
medicine with LAV in experimental JE
virus infection in adult Swiss albino mice
along with a preliminary observation on
this medicine s curative role also against
JE.
Kolkata, India. The medicine was prepared
by the company according to standard
procedures mentioned in Homeopathic
Pharmacopoeia of India (Ministry of
Health, Government of India, 1971, 1:1, 716, 72).
Procurement and storage of Japanese
Encephalitis Vaccine
The Japanese Encephalitis Live attenuated
Vaccine; (SA14-14-2) was kindly gifted
by
the State District Hospital,
Government. of West Bengal, India which
was maintained as per manufacturer s
instructions. The Government of West
Bengal (a state in India) originally
received the vaccine from Govt. of India
(Lot no-200811CO57-4) manufactured by
Chengdu Institute of Biological Products,
Chengdu, China.
The adult mice
In this experiment, adult Swiss albino
mice (Webster strain) were used after
getting permission from the Ethical
Committee of the Institute and maintained
in the mice colony of the School of
Tropical Medicine, Kolkata. In this
experiment the virulent Nakayama strain
(Source human, year 1935, location Japan,
GenBank accession no. EF571853,
Genotype III) was used.
Adult mice adaptation and inoculations
To study the effect of Belladonna 200
on JE virus infection in adult mice, the
experimental model was redesigned
mainly for preventive aspect study as all
previous experiments were mainly done on
suckling mice which succumb within 7296 hrs and immune-conversion is not
possible in suckling mice within this short
period of time. However, the real problem
Materials and Methods
The Medicine Belladonna 200
In this study we used Belladonna 200
which was procured directly from reputed
homeopathic drug company, Hahnemann
Publishing Co. Pvt. Ltd (HAPCO),
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Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 160-166
was the virus from infected suckling mice
brain does not affect the adult mice when
inoculated intracerebrally probably due to
absence of receptors, thickening of the
skull bone of adult mice etc. Thus initially
we established the JE virus infection by
gradual passage inoculation of the virus
obtained from infected suckling mice brain
in the adult mice (6 -8 wks). The virus was
consecutively passaged three times in mice
and the virus suspension was prepared
from the third stage which was designated
as the 10--1 stock suspension. For
determination of a 50% lethal dose (LD50),
several lots of mice (6 to 8 wks age) were
injected intracerebrally with dilutions of
the stock suspension from 10--1 to 10--9.
All the inoculated mice were observed
daily, their survival capacity was noted
and following this LD50 value was
calculated by the standard method
(Bonamin and Endler, 2010; Berdai et al.,
2012).
Experimental Group
Group 1 (Preventive aspect)
Randomly selected adult mice (6 in
number in each lot) were taken from litters
in which every mouse was orally fed with
0.06 mL of Belladonna 200 for 7 days.
The mice were challenged with LD50 (as
determined initially) JE virus suspension
(intra-cranially) after 21 days of the first
dose of administration of homoeopathic
medicine.
Group II (Vaccinated group)
Randomly selected in-bred adult mice (6
in number) in each lot) were initially
treated with two intra peritoneal doses of
0.03 ml of SA 14-14-2 JE vaccine on 0
and 7days. These mice were subsequently
challenged intra-cerebrally with LD50
dose (as determined initially) of the JE
virus suspension after 21 days of first dose
of vaccine.
After completing inoculations each mouse
was returned to the cage and was properly
labeled and kept in the rack. Mice showing
severe disease signs or those that died
within 2 h of observation were immersed
in a closed vessel containing chloroform
and later discarded according to statutory
guidelines
for
Biomedical
Waste
management.
Group III (Curative aspect)
Randomly selected in-bred adult mice (6
in number in each lot ) were initially
challenged intra-cerebrally with LD 50
dose (as determined initially) of the JE
virus suspension. These mice were
observed daily for signs of sickness. On
day 3 p.i. when mice showed signs of
sickness, they were treated with two doses
of 70 microl of Belledonna 200 at an
interval of one hour. The dose and dilution
of Belladonna 200, administered for
curative purposes was derived from the
references of Boenninghausen C.V (18431863), Boerick G (1960) and Farrington H
(1921). 60 microl of the medicine
(commercially obtained Bell 200) was
diluted with 10 microl of distilled water
Experimental design
Control Group
In control experiment, litters were not
orally fed with the medicine nor were they
vaccinated. These mice were only
challenged with intra-cerebral inoculation
of LD50 dose (as determined initially) of
JE virus.
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Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 160-166
and was shaken for 1 minute (Bradford
1921). 70 microl of the diluted medicine,
thus prepared, was administered orally as a
single dose to the JE infected mice. This
was followed by a second similar dose, 1
hr later. Thus only 2 doses of diluted
Belladonna 200 were administered to the
sick mice (at one hour interval) and they
were observed for a further period of 30
days.
Belladonna-200 43.4% mice died after
the JE virus challenge. An overwhelming
result was obtained in the vaccinated
group in which no mice died in the
experiment.
In the Belladonna-200
group in which the experimental mice
were treated in a preventive aspect (Table
1),
curative
aspect
(Table2)
or
simultaneously treated (Table 3) with the
virus inoculation the percentages of dead
mice were 13.6%, 22.8% and 50.0%
respectively. In statistical analysis 2 test
value in between the control group results
and the vaccinated group results was 22.19
and the similar value for the Belladonna200 group was 16.79. Both the 2 test
values were significant at 0.0005 level,
while the differences of the 2 test value in
between the vaccinated group and the
Belladonna-200 preventive group was
not statistically significant. Similarly if
we look into the 2 test value in the
curative group (Table 2), the difference is
not so significant between the control and
experimental group (the 2 test value only
3.60) and when we compare such values
between belladonna treated group with
vaccine treated group the 2 test value is
alsos not statistically significant. There is
no statistically significant difference
between
the
control
group
and
simultaneously treated group and the same
is true for the simultaneously Belladonna200 treated group.
Group IV (Simultaneously treated)
Randomly selected in-bred adult mice (6
in numbers in each lot) were initially
challenged intra-cerebrally with LD 50
dose (as determined initially) of the JE
virus suspension. These mice were then
immediately treated with two doses of 70
microl of Belledonna 200 at an interval of
one hour. The dose and preparation of the
medicine is as mentioned in Group III.
All the mice were observed daily after
inoculation and every four hours after the
onset of clinical signs. Clinical signs of the
disease in mice were refusal to feeds,
became disarranged in the nest, showed
tremors and muscular spasms, ataxia, and
hind-limb paralysis followed by death
within a few hours. Those mice that died
within the first 24 h were considered as
non-specific deaths. For preparation of
infected brain (adult adapted) suspension
required for LD50 determination and
standardization, the infected brains were
collected close to the time of death. In
curative treatment Belladonna-200 was
given in 3 days post JE virus inoculation
and in simultaneous treatment method
Belladonna-200
was
administered
simultaneously with virus inoculation.
Atropa Belladonna extract from which
ultradiluted Belladonna-200 is prepared
contains many bioactive chemicals like
hyoscyamine, atropine, hyoscine, apoatropine(atro-pamine),
belladonnine,
bellaradine and cuscohygrine. There are
also flavonoids; minerals like iron, copper,
cobalt, manganese which have also
different
biological
activities.
As
Belladonna-200 is prepared from crude
extract it is very difficult to predict which
Results and Discussion
In the control group in which the mice
were neither vaccinated nor treated with
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Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 160-166
Table.1 Shows the survival and death rate of JE infected adult mice (6 wks old) in preventive
treatment with Belladonna-200 for 7 days and compared with the control group and the
vaccinated group.
Group
Control
Vaccinated
Belladonna-200
Total no. of
inoculated mice
71
42
59
Number of
survived mice (%)
42 (59.15%)
42(100%)
51(86.4%)
Number of
died mice (%)
29 (40.84%)
00(00.0%)
08(13.6%)
Table.2 Shows the survival and death rate of JE infected adult mice (6 wks old) in curative
treatment (3 days post JE virus inoculation) with Belladonna-200 and compared with
control group and vaccinated group.
Group
Control
Belladonna-200
Total no. of
inoculated mice
36
36
Number of
survived mice (%)
19(52.77 %)
26(72.2%)
Number of
died mice (%)
17( 47.22 %)
10(22.8%)
Table.3 Shows the survival and death rate of JE infected adult mice (6 wks old) in
simultaneous treatment with Belladonna-200 and compared with control group and
vaccinated group.
Group
Control
Vaccinated
Belladonna-200
Total no. of
inoculated mice
12
06
06
is the key anti JE component of this
preparation. The whole plant is used for
the preparation of homeopathic drug
Belladonna-200 as per Homeopathic
pharmacopeia of India (HPI). Records of
activities of homeopathic preparation of
Belladonna has a long past record as back
as seventeenth and eighteenth centuries.
Thus it showed a good prophylactic power
in an epidemic of highly infectious scarlet
fever in Konigslutter during summer of
1799 when investigated by Dr.Samuel
Hahnemann,
a
renowned
German
Physician
and
medical
scientist
(Hahnemann, 1801). When he observed
that all the children who took a very small
dose of Belladonna in time remained
Number of
survived mice (%)
07(58.33 %)
01(16.66%)
03(50.0%)
Number of died
mice (%)
05( 41.66%)
05(83.33%)
03(50.0%)
impervious by this highly infectious
disease.
Later this finding was further strengthened
by the works of Bloch, Cramer, Velson,
Behr and others who administered this
medicine on thousands of patients during
epidemics.
Many physicians at that time adopted the
above-mentioned Hahnemann s protocol
for preventing the scarlet fever. According
to writings of Dr. Dudgeon (1820-1904),
10
conventional
physicians
used
prophylactic Belladonna on 1646 children
and only 123 (7.4%) were affected. These
excellent results of Belladonna revealed its
164
Int.J.Curr.Microbiol.App.Sci (2014) 3(1): 160-166
effectiveness in scarlet fever epidemic
when the rate of infection was 90% at that
time (Dudgeon 1853).
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Acknowledgement
We are grateful to the Registrar of West
Bengal University of Health Sciences to
accept this PhD research proposal. We are
also grateful to the Directors, School of
Tropical Medicine, Kolkata for their kind
permission to do this work in School of
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and other staff of Virology unit who
cordially helped us in our daily research
activities. We sincerely acknowledge Dr.
Rathin Chakraborty, Member, Scientific
Advisory Committee, CCRH, Government
of India, for his continuous cooperation in
this research. We are grateful to Prof P. N.
Gupta, Prof. Ranjit Mukherjee, Prof. S. K.
Mohiuddin and Prof. Nemai Bhattacharya
for their invaluable guidance in this study.
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